This invention relates to highly purified Giardia lamblia-specific stool antigen (GSA 65) and polyclonal monospecific antibodies specific for it. This invention further relates to diagnosis of human infection caused by Giardia lamblia.
Giardia lamblia is a protozoan parasite which inhabits the small intestines of humans. It is the most common cause of defined waterborne diarrhea in the United States, and massive outbreaks of diarrhea, particularly in children, have occurred due to Giardia-contaminated water supplies and person to person transmission. This affliction is referred to as giardiasis.
Giardiasis affliction has been diagnosed traditionally by microscope detection of cysts or trophozoites in stools or in material retrieved from the small intestine by invasive methods. Diagnosis of infection with Giardia lamblia by microscopic examination of stool for ova and parasites (O&P) is a laborious process. Even after the various standard methods of stool preparation which increase the sensitivity of Giardia detection are carried out, the sensitivity of O&P microscopic examination is dependent upon a microscopist's skill in scanning each preparation. The diagnostic success rate of stool examination is roughly 50-70%. Moreover, infectious cysts may not always be excreted despite giardial infection, necessitating multiple stool examinations which may not result in positive diagnosis.
In recent years, efforts have been made to improve the sensitivity of giardial diagnosis methods. The focus of these efforts has been primarily on serologic testing for anti-giardial antibodies and detection of giardial antigens in patient stool specimens. Serologic tests have proven to be of little value in giardial diagnosis because there is little correlation between positive anti-giardia antibody titers and the presence of active giardial infection. Cross-reactions with microbial antigens have also caused problems.
Stool antigen detection tests have been more successful. Craft, J. C., et al., J. Infect. Dis. 145:499-504 (1982) report the use of counterimmunoelectrophoresis (CIE) with rabbit antiserum prepared against G. lamblia cysts for detection of G. lamblia-specific antigens in stool. Vinayak, V. K., et al., Pediatr. Infect. Dis. 4:383-386 (1985) teach use of rabbit antiserum against trophozoites grown in culture in a CIE test for Giardia antigens in patients' stools. Ungar, L. P., et al., J. Infect. Dis. 149:90-97 (1984) describe use of rabbit and goat antisera prepared against trophozoites grown in culture in the development of an antigen-capture enzyme-linked immunosorbent assay (ELISA) for detecting Giardia antigens in stool. Green, E. L., et al., Lancet ii:691-693 (1985) similarly describe an antigen-capture ELISA using antisera prepared against trophozoites grown in culture as well as cysts. Similarly, Nash, T. E., et al., J. Clin. Micro. 25:1169-1171 (1987); Janoff, E. N., et al., J. Clin. Micro. 27:431-435 (1989); Stibbs, H. H., et al., J. Clin. Micro. 26:1665-1669 (1988) have described more recently immunoassays for detection of Giardia infection.
Despite good sensitivity, the above tests pose certain problems. First, such tests usually employ polyclonal polyspecific antibodies against the whole Giardia lamblia trophozoite and/or cyst, raising concerns of cross-reactivity with other gastrointestinal parasites. Further, most antigens targeted in these polyclonal polyspecific antibody based assays are labile to conventional laboratory fixatives and media used for collection, transport, and storage of stool specimens destined for O&P microscopic examination or stool culture. Such immunoassays thus require the use of untreated stool specimens and thus limitations are imposed upon existing stool collection procedures.
Identification of a Giardia lamblia-specific stool antigen (GSA 65) useful in the diagnosis of giardiasis has been reported previously, Rosoff, J. D., et al., J. Clin. Micro. 23(5):905-910 (1986) (I). The antigen was further characterized physically and chemically. Rosoff, J. D., et al., J. Clin. Micro. 24(6):1079-1083 (1986) (II). Notwithstanding the foregoing publications, the general procedures as described in these papers fail to teach certain key aspects of the protocol for obtaining GSA 65. These key steps, to be described in detail below, fully enable one of ordinary skill to isolate GSA 65 and produce monospecific polyclonal antibodies thereto. The disclosures of each of the above-identified publications are herein incorporated and made part of this disclosure subject to the acknowledged defects of both.